A variety of functionally diverse proteins are attached to the membranes of eukaryotic cells via inositol-containing glycophospholipids. One of the best studied of these proteins, the glycolipid-anchored variant surface glycoprotein (VSG) of the parasitic African trypanosome, Trypanosoma brucei, is indispensable to the survival of the parasite in its mammalian host and is an attractive target for the chemotherapeutic resolution of African sleeping sickness and animal trypanosomiasis in sub-Saharan Africa. Although a molecular definition of the glycolipid anchor is available for only a few proteins other than VSG, immunological studies show that many of the anchors are antigenically similar and therefore share common features. The structure of the glycolipids and their attachment to protein is unusual from several points of view and suggests the involvement of novel biosynthetic pathways in the assembly of the membrane anchor. The aim of this proposal is to elucidate the pathway of glycolipid anchor biosynthesis using trypanosomes as a model system. The experimental approach will exploit a cell-free system capable of synthesizing the newly identified precursor glycolipid, P2, and, an inositol-acylated form of P2 termed P3. Appropriate radiolabeled metabolites such as nucleotide sugars will be added to trypanosome membranes and lipid products will be identified by thin layer and column chromatography. Neutral glycans will be generated from purified lipids and total extracts and analyzed by a high-resolution HPLC method using the Dionex chromatography system. Using these methods, putative biosynthetic lipid intermediates and immediate donors of individual components of the glycolipid will be identified. Candidate lipid intermediates will be tested by adding back purified lipids to membranes and examining the products of continued synthesis and/or degradation. Similar experiments will be performed to determine the relationship between inositol-acylated lipids and their non-inositol- acylated counterparts. Sugar analogues and other inhibitors of glycosylation will be used to express a major surface protein with a hyperacylated glycolipid anchor, will be analyzed to examine developmentally regulated divergences in glycolipid anchor biosynthesis. These approaches will be used to arrive at a broad description of anchor assembly. Once this is available, topological probes of membrane structure will be used to examine the transmembrane synthesis and distribution of lipid intermediates. The structural similarities between the VSG anchor, the precursor glycolipids and other lipid anchors described thus far, suggest a uniform biosynthetic pathway. It is expected that an analysis of VSG glycolipid anchor synthesis will elucidate a pathway of general significance in eukaryotic cells.